Piotr P. Slonimski

RESPIRATION-DEFICIENT MUTANTS OF YEAST. II. BIOCHEMISTRY.

Abstract Investigations of various enzymic activities, respiratory capacity, and cytochrome content have been carried out on mutants of yeast which had: p genes resulting in the inability to utilize nonfermentable carbon sources for growth; the cy1 gene resulting in a partial deficiency of cytochrome c; the “loss” of the cytoplasmic factor (ϱ−) necessary for the synthesis of cytochromes a + a3 and b; and various combinations of these determinants. p4ϱ+ strains respired (but “ineffectually”) and had low concentrations of cytochromes a + a3 and b. p5ϱ+ strains were deficient in respiration and cytochromes a+a3. p1ϱ+, p6ϱ+, p7ϱ+, Pϱ− an all pϱ− strains were deficient in respiration and cytochromes a + a3 and b. Numerous strains, which had various alterations in the content of cytochromes a + a3, b, c, and c1 + b2, could be obtained by using various combinations of the p/P genes, cy1/CY gene and ϱ+/ϱ− cytoplasmic factor. The frequently occuring deficiency of cytochromes a + a3 and b, is discussed in relationship to mitochondrial structure.

Critical sequences within mitochondrial introns: Pleiotropic mRNA maturase and cis-dominant signals of the box intron controlling reductase and oxidase

We have established the DNA sequence of nine yeast mutants that prevent the expression either of the split cytochrome b gene alone (five mutants) or of two split genes, the cytochrome b gene and the cytochrome oxidase subunit I gene (four mutants). All the mutations analyzed are localized in intron 14 of the cob-box gene. We have extended the concept of the intron-encoded mRNA maturase, already described for intron 12, to the intron 14, and have adduced evidence that this box7 pleiotropic maturase is involved in the splicing of two distant gene transcripts. Such a process may constitute a regulatory mechanism that coordinates the expression of two structurally nonhomologous genes encoding two metabolically related enzymes. Analyses of cis-dominant mutations reveal the role of signal sequences in the recognition of the intron RNA sequences to be excised. These signal sequences are localized near the exon-intron boundaries (box1), or quite distant from the splicing sites, either in the blocked reading frame (box2) or in the open reading frame (box9) of the intron. We believe that for the last sequence, a ribosomal recognition of the box9 signal could be involved in a regulatory mechanism of the splicing of the pre-mRNA.

La synthèse adaptive des cytochromes chez la levure de boulangerie

1. n1. Bakers yeast, grown for a relatively short time in the absence of oxygen, cannot utilize glucose by respiratory mechanisms, but keeps its fermentative ability intact. At the same time it looses the cytochromes a, b and c, and acquires two new components analogous to cytochromes a1 and b1. n n2. n2. The synthesis of the normal cytochromes can be induced in such a yeast by oxygen in the absence of cell multiplication. Synthesis is accompanied by the disappearance of the new cytochrome components and results in the reestablishement of the ability to respire. n n3. n3. The mechanism of the adaptive synthesis of the cytochromes is discussed.

An mRNA maturase is encoded by the first intron of the mitochondrial gene for the subunit I of cytochrome oxidase in S. cerevisiae

We have localized ten oxi3- mutations in the first, al1, intron of the coxl gene. All are splicing deficient, being unable to excise the intron. Complementation experiments disclose several domains in the intron al1: the 5-proximal and 3-proximal domains harbor cis-dominant mutations, while trans-recessive ones are located in the introns open reading frame. Comprehensive analyses of allele-specific polypeptides accumulating in mutants show that they result from the translation of the introns ORF. We conclude that a specific mRNA maturase involved in splicing of oxidase mRNA is encoded by the intron al1 in a manner similar to the cytochrome b mRNA maturase.

Yeast Cells Lacking the Mitochondrial Gene Encoding the ATP Synthase Subunit 6 Exhibit a Selective Loss of Complex IV and Unusual Mitochondrial Morphology

Atp6p is an essential subunit of the ATP synthase proton translocating domain, which is encoded by the mitochondrial DNA (mtDNA) in yeast. We have replaced the coding sequence of Atp6p gene with the non-respiratory genetic marker ARG8m. Due to the presence of ARG8m, accumulation of ρ–/ρ0 petites issued from large deletions in mtDNA could be restricted to 20–30% by growing the atp6 mutant in media lacking arginine. This moderate mtDNA instability created favorable conditions to investigate the consequences of a specific lack in Atp6p. Interestingly, in addition to the expected loss of ATP synthase activity, the cytochrome c oxidase respiratory enzyme steady-state level was found to be extremely low (<5%) in the atp6 mutant. We show that the cytochrome c oxidase-poor accumulation was caused by a failure in the synthesis of one of its mtDNA-encoded subunits, Cox1p, indicating that, in yeast mitochondria, Cox1p synthesis is a key target for cytochrome c oxidase abundance regulation in relation to the ATP synthase activity. We provide direct evidence showing that in the absence of Atp6p the remaining subunits of the ATP synthase can still assemble. Mitochondrial cristae were detected in the atp6 mutant, showing that neither Atp6p nor the ATP synthase activity is critical for their formation. However, the atp6 mutant exhibited unusual mitochondrial structure and distribution anomalies, presumably caused by a strong delay in inner membrane fusion.

Yeast episome: Oligomycin resistance associated with a small covalently closed non-mitochondrial circular DNA

Abstract We have isolated a single step spontaneous mutant of S. cerevisiae resistant simultaneously to oligomycin, venturicidin, chloramphenicol, cycloheximide and triethyltin. This multiple drug resistance results from the interaction of two genetic factors showing both chromosomal location and episomal characteristics. One factor (π) confers oligomycin resistance, the other (τ) confers the other resistances. π can be lost spontaneously while τ can be completely eliminated with ethidium bromide. All π + strains, whether grande or petite, τ + or τ − , carry a covalently closed circular DNA while π − strains are devoid of it. We hypothesise that this circular DNA may play an informational role in the biogenesis and/or function of membranes.

Significance of Z-value statistics of Smith–Waterman scores for protein alignments

The Z-value is an attempt to estimate the statistical significance of a Smith-Waterman dynamic alignment score (SW-score) through the use of a Monte-Carlo process. It partly reduces the bias induced by the composition and length of the sequences. This paper is not a theoretical study on the distribution of SW-scores and Z-values. Rather, it presents a statistical analysis of Z-values on large datasets of protein sequences, leading to a law of probability that the experimental Z-values follow. First, we determine the relationships between the computed Z-value, an estimation of its variance and the number of randomizations in the Monte-Carlo process. Then, we illustrate that Z-values are less correlated to sequence lengths than SW-scores. Then we show that pairwise alignments, performed on quasi-real sequences (i.e., randomly shuffled sequences of the same length and amino acid composition as the real ones) lead to Z-value distributions that statistically fit the extreme value distribution, more precisely the Gumbel distribution (global EVD, Extreme Value Distribution). However, for real protein sequences, we observe an over-representation of high Z-values. We determine first a cutoff value which separates these overestimated Z-values from those which follow the global EVD. We then show that the interesting part of the tail of distribution of Z-values can be approximated by another EVD (i.e., an EVD which differs from the global EVD) or by a Pareto law. This has been confirmed for all proteins analysed so far, whether extracted from individual genomes, or from the ensemble of five complete microbial genomes comprising altogether 16956 protein sequences.

LARGE-SCALE PHENOTYPIC ANALYSIS : THE PILOT PROJECT ON YEAST CHROMOSOME III

In 1993, a pilot project for the functional analysis of newly discovered open reading frames, presumably coding for proteins, from yeast chromosome III was launched by the European Community. In the frame of this programme, we have developed a large‐scale screening for the identification of gene/protein functions via systematic phenotypic analysis. To this end, some 80 haploid mutant yeast strains were constructed, each carrying a targeted deletion of a single gene obtained by HIS3 or TRP1 transplacement in the W303 background and a panel of some 100 growth conditions was established, ranging from growth substrates, stress to, predominantly, specific inhibitors and drugs acting on various cellular processes. Furthermore, co‐segregation of the targeted deletion and the observed phenotype(s) in meiotic products has been verified. The experimental procedure, using microtiter plates for phenotypic analysis of yeast mutants, can be applied on a large scale, either on solid or in liquid media. Since the minimal working unit of one 96‐well microtiter plate allows the simultaneous analysis of at least 60 mutant strains, hundreds of strains can be handled in parallel. The high number of monotropic and pleiotropic phenotypes (62%) obtained, together with the acquired practical experience, have shown this approach to be simple, inexpensive and reproducible. It provides a useful tool for the yeast community for the systematic search of biochemical and physiological functions of unknown genes accounting for about a half of the 6000 genes of the complete yeast genome.

Functional analysis of RRD1 (YIL153w) and RRD2 (YPL152w), which encode two putative activators of the phosphotyrosyl phosphatase activity of PP2A in Saccharomyces cerevisiae.

Abstract In the context of the cooperative project for functional analysis of novel genes uncovered during the systematic sequencing of the Saccharomyces cerevisiae genome, we deleted two paralogous ORFs: YIL153w and YPL152w. Based on the resulting phenotypes, the corresponding genes were named RRD1 and RRD2, respectively. Rrd proteins show significant similarity to the human phosphotyrosyl phosphatase activator (PTPA). Both single mutants, rrd1Δ and rrd2Δ, were viable. Deletion of RRD1 caused pleiotropic phenotypes under a wide range of conditions, including sensitivity to Ca2+, vanadate, ketoconazole, cycloheximide and Calcofluor white, and resistance to caffeine and rapamycin. The only phenotypes found for rrd2Δ– resistance to caffeine and rapamycin – were weaker than the corresponding phenotypes of rrd1Δ. The double mutant rrd1,2Δ was inviable on rich glucose medium, but could grow in the presence of an osmotic stabilizer. The rrd1,2Δ mutant was partially rescued by inactivation of HOG1 or PBS2, suggesting an interaction between the RRD genes and the Hog1p signal transduction pathway. Introduction of slt2Δ into the rrd1,2Δ background improved the growth of rrd1,2Δ on sorbitol-containing medium, indicating that the Rrd proteins also interact with the Slt2p/Mpk1p signaling pathway. Suppression of the lethal phenotype of the rrd1,2Δ mutant by overexpression of PPH22 suggested that the products of the RRD genes function positively with catalytic subunits of PP2A. The synthetic lethality was also suppressed by the “viable” allele (SSD1-v1) of the SSD1 gene.

Chemotyping of yeast mutants using robotics.

By now, the EUROFAN programme for the functional analysis of genes from the yeast genome has attained its cruising speed. Indeed, several hundreds of yeast mutants with no phenotype as tested by growth on standard media and no significant sequence similarity to proteins of known function are available through the efforts of various laboratories. Based on the methodology initiated during the pilot project on yeast chromosome III (Yeast 13, 1547–1562, 1997) we adapted it to High Throughput Screening (HTS), using robotics. The first 100 different gene deletions from EUROSCARF, constructed in an FY1679 strain background, were run against a collection of about 300 inhibitors. Many of these inhibitors have not been reported until now to interfere in vivo with growth of Saccharomyces cerevisiae. In the present paper we provide a list of novel growth conditions and a compilation of 49 yeast deletants (from chromosomes II, IV, VII, X, XIV, XV) corresponding to 58% of the analysed genes, with at least one clear and stringent phenotype. The majority of these deletants are sensitive to one or two compounds (monotropic phenotype) while a distinct subclass of deletants displays a hyper‐pleiotropic phenotype with sensitivities to a dozen or more compounds. Therefore, chemotyping of unknown genes with a large spectrum of drugs opens new vistas for a more in‐depth functional analysis and a more precise definition of molecular targets. Copyright